Project Summary/Abstract Intrinsically disordered proteins and protein regions (IDPs and IDRs) lack stable tertiary structure but retain biological function. Understanding the structure/function relationships of such disordered protein regions presents a significant challenge because of their highly variable and dynamic nature. The past few years have resulted in an increased awareness and recognition of the prevalence and roles of IDPs and IDRs in membrane trafficking and organization. The primary goal of the proposed research is to advance our understanding of how the dynamic and highly variable structure of IDPs mediates their functions in membrane trafficking and organization. A key aspect of IDP function in membrane trafficking and organization involves direct IDP-membrane interactions, which can occur in conjunction with disorder-to-order transitions, or in the absence of protein ordering. Formation of membrane-binding amphipathic helices (AHs) is the most common example of the former, but the mechanisms underlying and regulating the formation, stability, specificity and function of such membrane-associated AHs remain poorly understood. Factors that govern membrane binding by IDRs that remain disordered in the bound state are even less well understood. A major area of proposed research centers on delineating mechanisms for these types of IDP-membrane interactions using the protein complexin as a model via a combination of in vitro characterization of structure and dynamics and in vivo functional assays. Another emerging aspect of IDP function is their ability to mediate the formation of condensates or membraneless organelles. Recently, it has been demonstrated that IDR-containing membrane- binding proteins can form cytosolic condensates that sequester and organize intracellular reservoirs of membrane vesicles. The mechanisms that regulate the ability of condensates to interact with and organize membranous vesicles or compartments have barely been explored and represent second major focus of this proposal. The primary model system for these efforts will be the clustering of membrane vesicles mediated by the protein synapsin, and the regulation of this clustering by IDPs and IDRs such as synucleins and rab proteins. Efforts will also include investigating the role of condensate formation in the organization of tubulo- vesicular organelles such as the endocytic recycling compartment (ERC). These systems will be characterized using structure/function analyses combining in vitro characterization of condensate formation and vesicle recruitment/release with in situ and in vivo functional studies. Achieving the overarching goals of this proposal will serve to advance our understanding of different mechanisms that underlie the roles of IDPs and IDRs in the regulation of membrane trafficking and organization. By focusing on specific models with physiological significance, namely factors governing vesicle exocytosis and the formation of clustered vesicular structures in neurons and other cell types, the results will make a significant impact on specific fields as well as broaden our general understanding of how protein disorder contributes to the organization of cellular membranes.